SARS-CoV-2 (COVID-19 Virus) Variant of Concern (VoC) S Screening and Genomic Sequencing for Surveillance

SARS-COV-2 VoC S-Gene Deletion Screen by Real-Time PCR

Due to the emergence of the Omicron (B.1.1.529) Variant of Concern, Public Health Ontario Laboratory, along with the COVID-19 Provincial Diagnostic Network, will implement enhanced surveillance for eligible COVID-19 positive specimens to assist in the early detection of potential cases in Ontario as of December 6, 2021.

The Omicron variant possesses a deletion at amino acid positions H69 and V70 within the spike protein. Thermo Fisher TaqPathTM PCR assay has significantly reduced S-gene target sensitivity, and is negative in the presence of this mutation. Thus a distinct result pattern know as S gene target failure (SGTF) is observed on the TaqPath real-time PCR platform.

SGTF is characterized by SARS-CoV-2 positive specimens that are S target negative, but positive for the two other targets in this assay, nucleoprotein (N) and open reading frame (orf1a/b). Based on current epidemiology, SARS-CoV-2 SGTF is consistent with Variant of Concern lineages B.1.1.529 (Omicron) or B.1.1.7 (Alpha), or other lineages, including Variants of Interest (VOIs), possessing this deletion. 

As of December 6, 2021, all COVID-19 PCR-positive specimens (with Ct ≤ 35) in Ontario will be screened for S gene target failure (SGTF) as a marker for the Omicron variant. 

  • Diagnostic labs who already use the TaqPathTM assay to detect SARS-CoV-2 will continue to do so and report SGTF via an add-on interpretation on health care practitioner reports. A proportion (up to 100% based on capacity) of SGTF detected SARS-CoV-2 specimens with Ct ≤ 30 from these labs will be forwarded to Ontario Covid-19 Genomics Network (OCGN) labs for whole genome sequencing.
  • Diagnostic labs that do not use the TaqPathTM assay will forward all SARS-CoV-2 positive specimens with a Ct ≤ 35 to OCGN labs for screening on the TaqPathTM assay. A proportion (up to 100% based on capacity) of these specimens with a Ct value of ≤ 30 from these labs will be forwarded for whole genome sequencing.

Table 1. SGTF VOC PHO Laboratory result interpretation.

Orf 1a/b gene N gene S gene Interpretation Additional Notes
Detected with Ct ≤ 30 Detected with Ct ≤ 30 Not Detected Result consistent with SARS-CoV-2 S gene target failure (SGTF) due to a deletion at amino acid positions 69-70. *
Detected Ct > 30 Detected Ct > 30 Not Detected Inconclusive for SARS-CoV-2 S gene target failure (due to low viral load) * The S gene may not bear the deletion at amino acids 69-70 but be Not Detected due to low viral load in the specimen.
Detected or Not Detected Detected or Not Detected Detected No evidence of SARS-CoV-2 S gene deletion at amino acid positions 69-70. *
One of Orf1a/b or N gene may be detected Or Neither Orf1a/b nor N gene is detected And S gene is not detected. Unable to complete Unable to complete SARS-CoV-2 VOC S gene deletion screening due to failure to detect one of the two additional gene targets in the assay (N or orf1ab) or failure to detect any of the 3 gene targets in the assay (S, N, orf1ab). Detection of both N and orf1ab gene targets is required for documentation of S gene target failure due to a deletion of amino acid positions 69-70. *
Failed to detect internal control Invalid Test results are uninterpretable due to failed detection of internal control(s). Please resubmit another specimen for testing if clinically indicated.

* Based on current epidemiology, SARS-CoV-2 S gene target failure (SGTF) is consistent with Variant of Concern (VOC) lineages B.1.1.529 (Omicron) or B.1.1.7 (Alpha), or other lineages, including Variants of Interest (VOIs), possessing this deletion.

All VOC S gene deletion screen PCR results (and subsequent sequencing results when applicable, see below) will be reported to the ordering healthcare provider; all VOC PCR results will be reported to the local Public Health Unit.

Ongoing VOC S gene deletion screen testing of COVID-19 positive specimens will be revisited as needed.

Persons with SARS-CoV-2-positive specimens displaying SGTF are considered persons under investigation (PUI) for Omicron VOC. See Ontario Ministry of Health: COVID-19 Variant of Concern Omicron (B.1.1.529): Case, Contact and Outbreak Management Interim Guidance for further information.  


SARS-CoV-2 Whole Genome Sequencing for Surveillance

As of November 12, 2021 PHO Laboratory will be randomly selecting a proportion (e.g. 25%) of all SARS-CoV-2 positive specimens, with Ct≤ 30* from the SARS-CoV-2 PCR diagnostic test, for whole genome sequencing (WGS).  This replaces the previous process implemented on May 26, 2021 for sequencing based on the VOC real-time PCR test.

The proportion of selected specimens will be adjusted periodically and determined according to positivity rates, the number of positive specimens, and testing capacity. When capacity allows, up to 100% of eligible specimens will be tested by WGS. This sampling method was first implemented on June 14, 2021, and aims to remove selection bias, provide better representation of known variants for surveillance, and allow improved detection of new emerging variants.

The following link provides the latest summary for SARS-CoV-2 Whole Genome Sequencing in Ontario.

In addition to the selected proportion (if less than 100%), sequencing will be performed on specimens meeting the following criteria:

  1. International travel – all specimens from travelers and contacts will be sequenced if travel is indicated on the COVID-19 diagnostic test requisition. If travel was not indicated on the requisition, testing can be added upon request.
  2. Vaccine breakthrough – all specimens from cases that are positive for SARS-CoV-2 ≥14 days after the completion of the vaccination series (e.g.  ≥14 days after the second dose of a 2-dose series) will be sequenced if vaccination status is indicated on the COVID-19 diagnostic test requisition. If vaccination status was not indicated on the requisition testing can be added upon request.
  3. Suspected reinfection – sequencing can be requested to support the classification of the case as re-infection.
  4. Certain outbreak investigations – sequencing can be requested to support outbreak investigations.

For criteria 1 and 2, sequencing results will be sent as a lab information system standard report back to the ordering healthcare provider and to the public health unit for all specimens, however only VOC lineages will be named on the report. Lineages not currently designated as VOC will be reported back as “Variant of Concern not detected” due to the dynamic nature of non-VOC lineage naming. Similarly, Variants of Interest (VOIs) will not be identified as such on the laboratory report, nor will the lineage be reported. Descendent lineages of VOCs will be reported as the parent lineage only (e.g. AY.4 Delta descendent lineage reported as B.1.617.2).

For criteria 3 and 4, outbreak investigations, the submitter may request a simple lineage-only report or in-depth genomic analysis.  If WGS testing is agreed to by the Ontario COVID-19 Genomic Network, submitters should confirm the specimen’s eligibility and availability with the original testing lab and arrange for specimens to be sent to the WGS testing laboratory. Please refer to the SARS-CoV-2 Whole Genome Sequencing Request guidance document and contact IORT@oahpp.ca for support. 

* The specimen’s SARS-CoV-2 PCR cycle threshold (Ct) value must be ≤ 30 for successful genome sequencing.  A subset of samples with a Ct ≤ 30 may not be successfully sequenced likely due to a low level of virus present, RNA degradation, or sequencing technical issues.

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit

SARS-CoV-2 Genomic Surveillance

SARS-CoV-2 PCR-positive specimen

1.5 ml preferred (min 1.0 ml)

Samples should be submitted in screw cap tube (Simport or O-ring, etc) only. Do NOT submit samples in snap cap

Submission and Collection Notes

1

For travel, vaccinated and super spreading event, if the specimen is at PHO Laboratory call Customer Service Centre (416-235-6556 or 1-877-604-4567) or fax the SARS–CoV-2 Variant of Concern Testing/Whole Genome Sequencing Requisition to (416-235-6552) to request to add the test; if the specimen is at another laboratory use the form above to send the specimen to PHO Laboratory.

2

For outbreak investigations email IORT@oahpp.ca to complete the request form and obtain approval/WGS-request ID before shipping any specimens. Submitters will receive information on signing up for a secure Incident and Outbreak Response Collaboration SharePoint Site account. This site has been authorized for the transfer of Personal Health Information and submitters will receive the in-depth genomic reports via this site.

Limitations

This laboratory-developed investigational assay is being used for surveillance purposes and public health management. It is not a clinical test.

Preparation Prior to Transport

For SARS-CoV-2-positive specimens being sent to PHO Laboratory for VOC mutation testing, 1.5 mL (minimum volume 1.0 mL) of specimen should be provided and maintained at 2-8oC during transportation (freeze if anticipated transport time to the laboratory is >72 hours).

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Surveillance testing will be performed at regular intervals. SARS-CoV-2 PCR assay to detect SARS-CoV-2 S gene target failure (SGTF) will be tested on specimens that meet acceptance criteria outlined above. Results will be available within 48 hours of receipt at PHOL.

If performed, SARS-CoV-2 genome sequencing results will be available within 2-4 weeks.

Reporting

All VOC PCR results (and subsequent sequencing results where applicable, see below) will be reported to the ordering healthcare provider; all VOC PCR results will be reported to the local Public Health Unit.

As a disease of public health significance, all SARS-CoV-2 whole genome sequencing results will be reported to the local public health unit and the ordering health-care provider as indicated on the requisition.

Test Methods

This laboratory-developed investigational assay is being used for surveillance purposes and public health management.

Sequencing involves PCR amplification, followed by sequencing of the majority of the approximately 30,000 base pair SARS-CoV-2 genome. Generated sequences are processed using bioinformatic tools and assigned a Pango lineage using the PANGOLINsoftware package, allowing for the identification of VOC and other lineages.

Algorithm

  1. A proportion of all SARS-CoV-2 positive specimens with Ct ≤ 30 will be selected for sequencing
  2. Specimens not already selected for sequencing that meet select conditions (e.g. post-vaccine, international travel, outbreak) can be requested to be sequenced
  3. As of December 6, 2021, specimens will also be selected for sequencing (WGS) based on results of the SGTF screened through Thermo Fisher TaqPathTM real-time PCR.

Data and Analysis

Updated 5 Dec 2021